An extensive description of the background and prior art for the diagnosis of gastric disease by the detection of H. pylori in gastric tissue is set forth in my co-pending U.S. patent application Ser. No. 08/195,954 and is incorporated herein above by reference.
Briefly, it having been seen that the bacteria H. pylori is present in endoscopically obtained gastric biopsy specimens from both gastric and duodenal ulcer patients and it being known that the enzyme urease is always associated with that bacteria, the concept of diagnosing the presence of such ulcers by testing biopsy specimens for urease suggested itself. Chemical tests for urease were already known in the art. In one such test a urea-containing broth provides a positive urease reaction (hydrolysis of urea) as below: EQU urea+H.sub.2 O urease.fwdarw.NH.sub.3 +CO.sub.2
as indicated by a change in color of the indicator Bacto phenol red from yellow (pH 6.8) to red to cerise at pH 8.1 or more alkaline due to the production of ammonia an&or ammonium carbonate by the urea-urease reaction. See the Difco Manual, 9th edition, Difco Laboratories, Detroit, Mich., (1953). The urea broth described in the Difco Manual was apparently used by B. J. Marshall in the work described in the Rapid Diagnosis of Campylobacteria associated with Gastritis, The Lancet, Jun. 22, 1985.
This type of urease test has come into commercial clinical use. In the United States a commercial test product is marketed under the trademark "CLOtest.RTM.". This product is described in U.S. Pat. No. 4,748,113 issued to Barry J. Marshall on May 31, 1988. The test of the Marshall patent commercially employs urea, a buffer, a bactericide, arid phenol red as the dye indicator. This test is carded out in an alkaline pH range showing a positive result on a change of the indicator from yellow to red at a pH in the range from about 6.8 to 9.
In the Marshall test, a gastric mucosal biopsy specimen containing H. pylori is placed in solution or an aqueous agar gel containing urea, and indictor, phenol red, and buffers. The urease in H. pylori converts to urea to ammonia which raises the pH and turns the agar color from a yellow to red, indicating a positive test. According to the package insert in the Marshall commercial phenol red test (CLOtest.RTM.)it is recommended that the test be incubated at 30.degree.-40.degree. C for three hours and it is indicated that it may take up to 24 hours to develop a positive test. This test relies on the passive diffusion of urease from the cell wall of the bacterium into the agar gel testing solution. Moreover, operating as it does at a pH above 6.5, the test may give a positive result with bacteria other than H. pylori and thus is not entirely specific for Helicobacter pylori. Specifically, Proteus, Pseudomonas, and E. Coli species may cause a color change at this level and give a false positive test.
Another test kit for H. pylori is available commercially from Serim Research Corporation, 1000 Randolph St., Bldg. 17, c/o Miles Inc., Elkhart, Ind. 56515 under the trademark "PyloriTek". This kit includes test strips having a substrate pad containing 3.3% urea and, in a separate matrix, a reaction pad containing 0.1% bromophenol blue dye indicator and 0.2% sulfamic acid. The test kit also contains, in a separate container, a hydration solution consisting of 1.8% Tris buffer. This kit, makes use of the same urease-urea reaction as the Marshall test to produce gaseous ammonia which changes the bromophenol blue from its original yellow color to make a blue test spot over a biopsy specimen on a yellow field to indicate a positive test. This test is said to be readable in 120 minutes and should not be read after that time to avoid false positives. While the two-hour usual test time is an improvement, it is apparent that an even more rapid and less complicated test would be desirable.